title: The dimerization domain in outer segment guanylate cyclase is a Ca²⁺-sensitive control switch module.
abstract: Membrane-bound guanylate cyclases harbor a region called the dimerization or linker domain, which aids the enzymes in adopting an optimal monomer-monomer arrangement for catalysis. One subgroup of these guanylate cyclases is expressed in rod and cone cells of vertebrate retina, and mutations in the dimerization domain of rod outer segment guanylate cyclase 1 (ROS-GC1, encoded by the GUCY2D gene) correlate with retinal cone-rod dystrophies. We investigate how a Q847L/K848Q double mutation, which was found in patients suffering from cone-rod dystrophy, and the Q847L and K848Q single-point mutations affect the regulatory mechanism of ROS-GC1. Both the wild type and mutants of heterologously expressed ROS-GC1 were present in membranes. However, the mutations affected the catalytic properties of ROS-GC1 in different manners. All mutants had higher basal guanylate cyclase activities but lower levels of activation by Ca²⁺-sensing guanylate cyclase-activating proteins (GCAPs). Further, incubation with wild-type GCAP1 and GCAP2 revealed for all ROS-GC1 mutants a shift in Ca²⁺ sensitivity, but activation of the K848Q mutant by GCAPs was severely impaired. Apparent affinities for GCAP1 and GCAP2 were different for the double mutant and the wild type. Circular dichroism spectra of the dimerization domain showed that the wild type and mutants adopt a prevalently α-helical structure, but mutants exhibited lower thermal stability. Our results indicate that the dimerization domain serves as a Ca²⁺-sensitive control module. Although it is per se not a Ca²⁺-sensing unit, it seems to integrate and process information regarding Ca²⁺ sensing by sensor proteins and regulator effector affinity.
authors.affiliation: Biochemistry Group, Department of Neurosciences, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Zägel
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D020013:Calcium Signaling; D002417:Cattle; D019281:Dimerization; D004795:Enzyme Stability; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D050505:Mutant Proteins; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C110715:guanylate cyclase 1; D006162:Guanylate Cyclase